Flow cytometry

Expression of Nectin4 on the surface of tumor cells was detected by anti-human Nectin4 Alexa Fluor^® 647-conjugated antibody (Clone #337516, R&D Systems, USA); Nectin4 CAR and Nectin4-7.19 CAR expression was detected by the fusion protein of Nectin4 extracellular domain with streptavidin (Nectin4-streptavidin), and then followed by the anti-streptavidin antibody with PE fluorescein (Clone #3A20.2). Expression of FAP was detected by anti-human FAP PE (Clone #427819, Bio-techne); FAP CAR expression was detected by anti-mouse IgG (H+L), Biotinylated Antibody (Cat #14709, Cell Signaling Technology, USA) and followed by streptavidin-tagged APC. Then, CAR-T cells were collected and their phenotype was assessed with anti-human CD4 PE/Cy7 (Clone: OKT4, Dilution: 1/400), anti-human CD8a Pacific Blue™ (Clone: RPA-T8), anti-human CD45RO Alexa Fluor^® 488 (Clone: UCHL1), anti-human CD45RA APC (Clone: HI100), and anti-human CCR7 PerCp/Cy5.5 (Clone: G043H7). For analysis of immunological checkpoints, the following antibodies were used: anti-human TIM3 Alexa Fluor^® 647 (Clone: 7D3, BD Biosciences), anti-human LAG3 PE (Clone: T47-530, BD Biosciences, USA), anti-human PD-1 APC (Clone: EH12.2H7), and anti-human CTLA-4 APC (Clone: L3D10). mCAR-T cells were labeled with anti-mouse CD8 PE (Clone: 53-6.7) and anti-mouse CD4 PE (Clone: RM4-5, Mutiscience, Hangzhou, CN), fixed and permeabilized with the LIVE/DEAD Fixable Aqua Dead Cell Stain kit (Invitrogen, CN), and labeled with anti-mouse IFN-γ APC (Clone: XMG1.2, Dilution: 1/50, eBioscience, Wuhan, CN) for intracellular staining. All antibodies of brands not mentioned above were from BioLegend and dilutions not mentioned above were 1/200. The isotype-matched IgG1 was used as a negative control. Cells were analyzed by a FACS Aria IIFlow Cytometer (BD Biosciences). Data were analyzed with FlowJo 10 (FlowJo, USA).